Measuring levels of a metabolite

ABSTRACT

Described herein are methods for determining an amount of an analyte in a test sample. The methods involve preparing a calibration curve using standard samples containing an isotopically-labeled standard in a biological matrix.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/843,588, filed on Jul. 26, 2010, which in turn claims priority toU.S. Provisional Application Ser. No. 61/228,444, filed on Jul. 24,2009. Each of these applications is incorporated by reference in itsentirety.

TECHNICAL FIELD

This document relates to methods and materials for determining an amountof an analyte in a sample using a mass spectrometry technique.

BACKGROUND

Mass spectrometry is an important analytical technique for determiningthe levels of many clinically relevant analytes in patient samples.Vitamin D metabolites and steroids are commonly tested examples ofclinically relevant analytes for assessing the health state ofindividuals, from birth through adulthood. Improving the accuracy ofmeasuring such analytes using mass spectrometry would allow for betterdetection of adverse health conditions.

SUMMARY

Described herein are methods for determining an amount of an analyte ina test sample. The methods involve preparing a calibration curve usingstandard samples containing an isotopically-labeled standard in abiological matrix.

For example, provided herein is a method for determining an amount of ananalyte in a test sample. The method includes:

-   -   (a) providing a set of calibrator samples, each calibrator        sample comprising a different amount of a first        isotopically-labeled standard in a biological matrix,    -   (b) providing to each calibrator sample a selected constant        amount of a second isotopically labeled standard, the first and        second isotopically labeled standards having distinct isotope        content;    -   (c) subjecting the calibrator samples comprising the first and        second isotopically-labeled standards to mass spectrometry to        determine signal intensities corresponding to the first and        second isotopically-labeled standards;    -   (d) generating a calibration curve based on ratios of the        determined signal intensities of the first and second        isotopically-labeled standards as a function of the amount of        the first isotopically-labeled standard in the calibrator        samples;    -   (e) contacting a test sample with the selected constant amount        of the second isotopically-labeled standard;    -   (f) subjecting the test sample comprising the second        isotopically-labeled standard to mass spectrometry to determine        signal intensities corresponding to the second isotopically        labeled standard and the analyte, and    -   (g) using the calibration curve and the determined signal        intensities corresponding to the second isotopically labeled        standard and the analyte to determine the amount of the analyte        in the test sample.

An analyte for use the methods described herein can include asecosteroid metabolite and a steroid metabolite. The secosteroidmetabolite can be, for example, 25-hydroxyvitamin D3, 1-hydroxyvitaminD3, 1, 25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, 1α-hydroxyvitaminD2, and 24-hydroxyvitamin D2. For example, the analyte can be a vitaminD3 metabolite and the first isotopically-labeled standard can beselected from isotopically labeled 1, 25-dihydroxyvitamin D3 andisotopically-labeled 25-hydroxyvitamin-D3. The firstisotopically-labeled standard can be labeled with ²H, ¹³C, ¹⁵N, ¹⁸O, ora combination thereof. In some cases, the first isotopically-labeledstandard is selected from labeled 1α,25-dihydroxyvitamin D3, d₆-labeled25-hydroxyvitamin-D3, d₃-labeled 1, 25-dihydroxyvitamin D3 andd₃-labeled 25-hydroxyvitamin-D3. For example, the firstisotopically-labeled standard can be 25-hydroxyvitamin-D3(26,26,26,27,27,27-d6).

In some embodiments, the analyte can be a vitamin D3 metabolite and thesecond isotopically-labeled standard is selected from isotopicallylabeled 1, 25-dihydroxyvitamin D3 and isotopically-labeled25-hydroxyvitamin-D3, such as d₆-labeled 1, 25-dihydroxyvitamin D3,d₆-labeled 25-hydroxyvitamin-D3, d₃-labeled 1, 25-dihydroxyvitamin D3 ord₃-labeled 25-hydroxyvitamin-D3. For example, the secondisotopically-labeled standard is 25-hydroxyvitamin-D3 (6,19,19-d3).

The analyte can also be a vitamin D2 metabolite. In some embodiments,where the analyte is a vitamin D2 metabolite, the firstisotopically-labeled standard is selected from 1, 25-dihydroxyvitamin D2and 25-hydroxyvitamin-D2. For example, the first isotopically-labeledstandard can be 25-hydroxyvitamin-D2 (26,26,26,27,27,27-d6). In somecases, when the analyte is a vitamin D2 metabolite, the secondisotopically-labeled standard is selected from 1, 25-dihydroxyvitamin D2and 25-hydroxyvitamin-D2. For example, the second isotopically-labeledstandard can be 25-hydroxyvitamin-D2 (6,19,19-d3).

A steroid, as provided herein, can be selected from 11-deoxycortisol,21-deoxycortisol, 17-α-hydroxyprogesterone, aldosterone,4-androstene-3,17-dione, corticosterone, deoxycorticosterone, cortisol,dehydroepiandrosterone, dehydroepiandrosterone sulfate, estradiol,estriol, estrone, progesterone, 5-α-dihydrotestosterone, testosterone,pregnenolone, 17-α-hydroxypregnenolone, 5-α-androstane-3b,17b-diol,2-hydroxyestradiol, 4-hydroxyestradiol, high-density lipoprotein (HDL)cholesterol, low-density lipoprotein (LDL) cholesterol, and verylow-density lipoprotein (VLDL) cholesterol, and the first and secondisotopically labeled standards can be isotopically-labeled analogs ofthe selected analyte. For example, the analyte can be testosterone andan isotopically-labeled standard can be testosterone (3,4-¹³C;2,2,4,6,6-d5); the analyte can be 4-androstene-3,17-dione and anisotopically-labeled standard can be 4-androstene-3,17-dione (3,4-¹³C;2,2,4,6,6-d5); the analyte can be dehydroepiandrosterone and anisotopically-labeled standard can be dehydroepiandrosterone (3,4-¹³C;2,2,3,4,4,6-d5); and the analyte can be progesterone and theisotopically-labeled standard can be progesterone (3,4-¹³C;2,2,5,6,6,17a,21,21,21-d9).

The methods described herein can use any mass spectrometry method,including, for example, LC-MS and LC-MS/MS. In some embodiments, thetest sample is a biological sample. For example, the biological samplecan be selected from the group consisting of blood (e.g., blood provideddried on a paper matrix), serum, plasma, saliva, and urine.

The first and second isotopically-labeled standards have distinctisotope content. In some embodiments, the first and secondisotopically-labeled standards each comprise a different number ofisotopic labels comprising the same isotope.

Further provided herein is a kit for determining an amount of an analytein a test sample, comprising: an isotopically labeled calibratorstandard, and an isotopically labeled internal standard, wherein thecalibrator and internal standards have distinct isotope content.

DESCRIPTION OF DRAWINGS

FIGS. 1(A) and (B) show calibration curves for exemplary standardcompound d⁶-25-hydroxyvitamin D3.

FIGS. 2 (A) and (B) show calibration curves for exemplary standardcompound d⁶-25-hydroxyvitamin D2.

FIG. 3 shows exemplary isotopically labeled compounds.

FIG. 4 shows detection of residual analytes present in samples after astripping procedure was performed: (A) testosterone, (B) progesteroneand (C) a vitamin D metabolite.

DETAILED DESCRIPTION

Described herein are methods for determining an amount of an analyte ina test sample. The methods involve preparing a calibration curve usingstandard samples containing an isotopically-labeled standard in abiological matrix.

In typical clinical laboratory procedures, external calibration curvesusing standard solutions of the compounds of interest are employed forthe quantitation of the analytes in a test sample. These calibrationcurves cover the clinical ranges for the analytes of interest and aretypically prepared in matrices that mimic as much as possible thebiological samples used in the assay. In order to achieve the clinicalranges, the matrices (e.g. serum) used in the manufacturing of externalcalibrators are stripped from the endogenous analytes naturally presentin them. Most commonly, serum or plasma matrices are processed usingcharcoal or resin columns. When the matrices travel through thesecolumns, the charcoal or resin captures organic compounds present in thematrices. These stripping techniques are generally successful inremoving a good number of small organic compounds. However, they are noteffective in removing all natural analytes present in the matrix. Forexample, charcoal or resin stripping are not effective in removingvitamin D and related metabolites from serum and plasma matrices. As isdescribed in the Examples herein, provided are external calibrators thatare not affected by the presence of un-stripped endogenous analytes andthus provide the ability of constructing external calibration curvesthat range from true zero to high clinical and analytical ranges.

The role of a calibration curve is to permit accurate measurement of thelevel of an analyte in a sample. To generate a calibration curve, aseries of calibrator samples having increasing concentrations of acalibrator, in this case an isotopically-labeled analyte derivative, forexample, an isotopically-labeled vitamin D metabolite, as describedherein in the Examples, are subjected to a mass spectrometry techniquewhere one or more mass spectrometry signals of the calibrator aremeasured. As described herein, a linear calibration curve can bedeveloped by determining a mass spectrometry signal corresponding to theamount of calibrator in each standard sample. For clinical testing, thestandard samples are often biological samples that have been depleted ofendogenous analyte. For example, standard samples for testing vitamin Din blood are blood serum samples that have been charcoal stripped.However, there is often an amount of analyte remaining, in this casevitamin D metabolites. By using the isotopically-labeled vitamin Dmetabolite as a calibrator, as described herein, the user can develop alinear multi-point calibration curve with a true zero point. The abilityto determine a true zero point is useful for detecting and accuratelymeasuring the level of an analyte present in a biological sample. Massspectrometry can be used to detect and measure the signal intensities(e.g., peak height) of the analyte and, if desired, peak height ratiosof the analyte and an internal standard can be used to determine amountof the analyte in each test sample by relating an analyte/internalstandard signal ratio from the test sample to the calibration curveexpressing a calibrator/internal standard ratio having a true zeropoint.

A calibration curve can be generated using a set of calibrator samples,each sample having a different amount of a first isotopically-labeledstandard. To each of these samples, the same amount of a secondisotopically labeled standard (i.e., an internal standard) can be added.The first and second isotopically-labeled standards can have distinctisotopic labeling. As described herein, a linear calibration curve canbe developed by determining a mass spectrometry signal corresponding tothe first standard and second standard in each calibrator standardsample and generating a calibration curve based on the ratios of thesignal intensities of the first and second isotopically-labeledstandards as a function of the amount of the first isotopically-labeledstandard in the calibrator samples. When determining the amount ofanalyte in the test sample, the second isotopically labeled standard(i.e. internal standard) is added to the sample in the same amount as ispresent in the calibrator samples, and mass spectrometry can be used todetect and measure the signal intensities (e.g., peak height) of ananalyte and the second isotopically-labeled standard in a test sampleUsing the calibration curve and the determined signal intensitiescorresponding to the second isotopically-labeled standard and theanalyte, the level of the analyte present in a biological sample ismeasured.

A series of standard calibration standard samples (e.g., 3, 4, 5, 6, 7,8, 9, and 10 or more standard samples) containing anisotopically-labeled calibrator standard at increasing concentrationscan be prepared. Several series having different calibratorconcentration ranges can be used. Concentrations can be selected on thebasis of the concentration range expected for a particular analyte andsample source. For example, a series of standard concentrations forvitamin D can include 5, 10, 20, 40, 75, and 150 ng/mL calibratorconcentrations. Calibration curve standard samples can be prepared andanalyzed using multiple replicates, for example, in duplicate ortriplicate.

A calibrator is an isotopically-labeled derivative of the analyte ofinterest and can have a distinct mass and, thus, distinct mass to charge(m/z) ratio from that of the analyte. Any appropriate isotopic label canbe used such as, for example, ²H, ¹³C, ¹⁵N, or ¹⁸O. For example, if theanalyte of interest is the vitamin D metabolite 1α,25-dihydroxyvitaminD3, the calibrator can be an isotopically-labeled derivative of themetabolite such as d₆-1α,25-dihydroxyvitamin D3. In some cases, specificcalibrators can include 25-hydroxyvitamin-D3 (26,26,26,27,27,27-d⁶) or25-hydroxyvitamin-D2 (26,26,26,27,27,27-d⁶). The isotopically-labeledvitamin D metabolites described herein are virtually chemicallyidentical to their non-labeled counterparts except that their molecularmass differs by six daltons. This difference in mass allows a massspectrometer to differentiate between the isotope-labeled vitamin Dmetabolites from the endogenous non-labeled metabolites present in thematrix. Likewise, if the analyte of interest is testosterone, thecalibrator can be an isotopically-labeled derivative of testosterone,such as that shown in FIG. 3.

Preparation of calibrator standards and test samples can also includeadding a constant, known concentration of one or more internalstandards, referred to as a second isotopically labeled standard herein,to a sample to allow for quantitation of the analyte of interest.Generally an internal standard is used when performing quantitationusing a mass spectrometry technique. This standard serves as a controlfor loss of analyte during sample preparation and instrument injection,and ion variability. An internal standard is generally added prior tosample preparation and analysis, and is added at the same level in everysample including the test sample and calibrator standards. The amount ofinternal standard used is above the limit of quantitation by theselected mass spectrometry technique but not so high as to suppressionization of the analyte. Often the amount of internal standard used istargeted to be in the lower one third of the working calibration curve.An internal standard useful in the methods described herein can beisotopically labeled. One or more isotopic labels can be used, and whenmore than one is used, multiple of the same label (e.g. deuterium) ordifferent labels (e.g. deuterium and ¹³C) can be present. Internalstandards can also be added to a test sample to distinguish naturallyoccurring (endogenous) molecules. In some cases, test sample preparationcan involve mixing a sample (e.g., a blood sample) with an extractionsolution in which one or more internal standards have been added.Alternatively, the internal standards can be added to a mixture of abiological sample and an extraction solution at any step in the samplepreparation that ensures the internal standards will not be removed fromthe mixture during the sample processing (e.g., after a liquid-liquidextraction or a solid phase extraction). In other cases, the internalstandards can be added to the test sample in the absence of anextraction solution. In some cases, test sample preparation can includeequilibration of the sample and one or more internal standards for aperiod of time (e.g., 5, 10, 15, 20, 25, 30, 60, 120, or more minutes).The equilibration temperature can be from about 10° C. to about 45° C.,or any value in between (e.g., 15, 25, 30, 35, 37, 42, or 44° C.). Insome cases, equilibration can be at room temperature for about 15minutes.

An internal standard can be any compound that would be expected tobehave under the sample preparation conditions in a manner similar tothat of one or more of the analytes of interest. Typically an internalstandard can be an isotopically-labeled derivative of the analyte ofinterest but could also be any appropriate analyte analog that isdetectable by mass spectrometry and distinguishable from the analyte ofinterest and the calibrator by its mass to charge ratio. Exemplaryisotopically-labeled internal standards are those derivatives that canbe clearly differentiated from the isotope peaks of the analyte ofinterest. Any appropriate isotopic labels can be used including, forexample, ²H, ¹³C, ¹⁵N and ¹⁸O or combination thereof. While not beingbound by any theory, the physicochemical behavior of such stableisotopically-labeled derivative with respect to sample preparation andsignal generation would be expected to be identical to that of theunlabeled analyte, but clearly differentiable on the mass spectrometer.In some cases, an internal standard can be deuterated 25-hydroxyvitaminD₂ or deuterated 25-hydroxyvitamin D₃. For example, d₆-labeled25-hydroxyvitamin D2; d₆-labeled 25-hydroxyvitamin D3, d₆-labeled1,25-dihydroxyvitamin D2; and d₆-labeled 1,25-dihydroxyvitamin D3 can beused. In some cases, an internal standard can be 25-hydroxyvitamin-D3(26,26,26,27,27,27-d6) or 25-hydroxyvitamin-D2 (26,26,26,27,27,27-d6).These compounds are also suitable calibrator standards.

In some embodiments, an isotopically-labeled internal standard orcalibrator standard can be a labeled steroid. For example, testosterone(3,4-¹³C;2,2,4,6,6-d5); 4-androstene-3,17-dione (3,4-¹³C;2,2,4,6,6-d5),dehydroepiandrosterone (3,4-¹³C;2,2,3,4,4,6-d6); and progesterone(3,4-¹³C;2,2,5,6,6,17a,21,21,21-d9).

Test samples appropriate for the methods described herein include anybiological fluid, cell, tissue, or fraction thereof, containing orsuspected of containing an analyte of interest. A test sample can be,for example, a specimen obtained from an individual (e.g., a mammal suchas a human) or can be derived from such an individual. For example, atest sample can be a biological fluid specimen such as blood, serum,plasma, urine, lachrymal fluid, and saliva. A test sample can also be atissue section obtained by biopsy, or cells that are placed in oradapted to tissue culture. Additional exemplary test samples includecultured fibroblasts, cultured amniotic fluid cells, chorionic villussample, skin sample, hair sample and the like. A test sample can befurther fractionated, if desired, to a fraction containing particularcell types. For example, a blood sample can be fractionated into serumor into fractions containing particular types of blood cells such as redblood cells or white blood cells (leukocytes). If desired, a test samplecan be a combination of samples from an individual such as a combinationof a tissue and fluid sample, and the like. Methods for obtaining testsamples that preserve the activity or integrity of molecules in thesample are well known to those skilled in the art. Such methods includethe use of appropriate buffers and/or inhibitors, including nuclease,protease and phosphatase inhibitors, which preserve or minimize changesin the molecules in the sample. Such inhibitors include, for example,chelators such as ethylenediamne tetraacetic acid (EDTA), ethyleneglycol bis(Paminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA), proteaseinhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin,leupeptin, antipain and the like, and phosphatase inhibitors such asphosphate, sodium fluoride, vanadate and the like. Appropriate buffersand conditions for isolating molecules are well known to those skilledin the art and can be varied depending, for example, on the type ofmolecule in the sample to be characterized (see, for example, Ausubel etal., Current Protocols in Molecular Biology (Supplement 47), John Wiley& Sons, New York (1999); Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane,Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999);Tietz, Textbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds.W.B. Saunders, Philadelphia, (1999)).

In some cases, a test sample can be processed to eliminate or minimizethe presence of substances which may interfere with the massspectrometry technique. For example, prior to performing massspectrometry, a test sample can be separated using standard techniques,such as by centrifugation, chromatography, binding to a matrix such as afilter paper, and extracting using extraction solution(s). A variety oftechniques can be used according to the sample type. For example, solidand/or tissue test samples can be ground and extracted to free theanalyte of interest from interfering components. A test sample can becentrifuged, filtered, and/or subjected to chromatographic techniques toremove interfering components (e.g., proteins, cells or tissuefragments). Hormonal steroids and their analogs and metabolites(compounds produced by biological transformation) can be sensitive tostrong acids or alkaline solution and often are treated under neutralconditions for isolation. Some steroidal analytes are acidic and can beheld in alkaline solution, while non-acidic impurities can be extractedwith organic solvents. In some cases, reagents known to precipitate orbind the interfering components can be added to a test sample. Forexample, whole blood samples can be treated using conventional clottingtechniques to remove red and white blood cells and platelets. Anyappropriate method of polypeptide extraction or precipitation can beperformed to deplete high abundance and high molecular weightpolypeptides from a biological sample (e.g., plasma, urine) prior tomass spectrometric analysis. For example, acetonitrile polypeptideextraction/precipitation can be performed. In some cases, potassiumhydroxide (KOH) or sodium hydroxide (NaOH) can be used for proteinextraction/precipitation, optionally followed by centrifugation of thetest sample. In some cases, immunochemistry-based protein-depletiontechniques can be performed to remove high abundance proteins from abiological sample. For example, commercially-available kits such as theProteoPrep® 20 (Sigma-Aldrich) plasma immunodepletion kit can be used todeplete high abundance proteins from plasma.

For use in the methods provided herein, a test sample can be in avariety of physical states. For example, a test sample can be a liquidor solid, dissolved or suspended in a liquid, in an emulsion or gel, orabsorbed onto a substrate, such as a paper or polymer substrate.

The amount of analyte in a test sample can be measured according to themethods provided herein. A calibration curve can be developed byanalyzing the differently labeled calibrator and internal standard usinga mass spectrometry technique. Test samples can be subjected to a massspectrometry technique to measure one or more mass spectrometry signalsfor the analyte of interest and an isotopically-labeled internalstandard. The peak height ratios of the analyte and internal standardcan be calculated and converted into measurements of the amount of theanalyte in each test sample by comparing the analyte/internal standardsignal ratio from the test sample to the calibration curve. The inverseratios and other mathematical expressions also can be used so long asthe amount of analyte detected in the test sample is determined.

Mass spectrometry analysis can be conducted with a single mass analyzer(MS), a “tandem in space” analyzer such as a triple quadrupole tandemmass spectrometer (MS/MS) and other MS configurations. As an example,tandem mass spectrometry can be used to distinguish and/or measure morethan one isomer in a single sample with one analysis. For MS/MS, twomass analyzers are linked in series via a collision cell. The first massanalyzer (MS-1) is used to select one or more of the molecular ions ofthe analyte of interest (e.g., an ion of a particular mass-to-chargeratio (m/z)) or of the internal standard or of the calibrator. Theselected ions are then transferred to a collision cell where they arefragmented by collisions with an inert gas. This process is calledcollisionally activated dissociation (CAD). Once the parent (sometimesreferred to as precursor) ions have fragmented, the second mass analyzer(MS-2) is used to either, scan and detect all of the produced daughter(sometimes referred to as product) ions or to select and detectparticular fragment ions.

The above-described mass spectrometry technique can also be referred toas multiple reaction monitoring (“MRM”). In MRM, a parent ion ofinterest is selected in MS-1, fragmented in the collision cell and aspecific fragment ion resulting from the collisional activation isselected in MS-2 and finally detected. MS-1 and MS-2 are fixed torespectively select the corresponding parent and fragment ion pairs ofinterest for a predetermined amount of time (e.g., a few milliseconds).This specific parent ion-product ion transition can be considered as onedetection channel. If additional analytes are to be detected, additionaldetection channels with specific mass transitions can be introduced inthe experiment. Data from all selected mass transitions (channels) canbe acquired sequentially to obtain the desired information.

Any tandem mass spectrometry instrument, including LC-MS and LC-MS/MSinstruments can be used. Exemplary tandem mass spectrometers areavailable from: PerkinElmer, Waters Corporation, Thermoelectron, andSciex. Commonly used tandem mass spectrometers are electrospray triplequadrupoles. An exemplary specific model is the ABI 4000 triplequadrupole tandem mass spectrometer (ABI-SCIEX, Toronto, Canada).Software for tuning, selecting, and optimizing ion pairs is alsoavailable, e.g., Analyst Software Ver. 1.4 (ABI-SCIEX).

An analyte to be measured according to the methods provided herein isdetectable by a mass spectrometry technique. In some cases, analytes canbe metabolites of the secosteroid vitamin D. Secosteroids such asvitamin D are similar in structure to steroids with the exception thattwo of the B-ring carbon atoms (C9 and 10) of the typical four steroidrings are not joined. For example, the analyte of interest can be25-hydroxyvitamin D2 (also known as ergocalciferol), 25-hydroxyvitaminD3 (also known as calciferol or calcidiol), 1α,25-dihydroxyvitamin D3(also known as calcitriol or 1α,25-dihydrocholecalciferol);1α-hydroxyvitamin D2, 1α-hydroxyvitamin D3, or 24-hydroxyvitamin D2. Insome cases, an analyte of interest can be a steroid or other metabolicintermediate. Steroids are terpenoid lipids characterized by a carbonskeleton with four fused rings, generally arranged in a 6-6-6-5 fashion.Steroids appropriate for the methods and materials provided herein caninclude, without limitation, 11-deoxycortisol; 21-deoxycortisol;17-alpha-hydroxyprogesterone; aldosterone; 4-androstene-3,17-dione;corticosterone; deoxycorticosterone; cortisol; dehydroepiandrosterone;dehydroepiandrosterone sulfate; estradiol; estriol; estrone;progesterone; 5-alpha-dihydrotestosterone; testosterone; pregnenolone;17-alpha-hydroxypregnenolone; 5-alpha-androstane-3b,17b-diol;2-hydroxyestradiol; and 4-hydroxyestradiol. In some cases, an analyte ofinterest can be high-density lipoprotein (HDL) cholesterol, low-densitylipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL)cholesterol, glucose, creatine, triglycerides, bilirubin, anelectrolyte, or amylase.

Information collected according to the methods provided herein can beused to assess the health state of a mammal (e.g., a human patient),such as presence or absence of a disorder (e.g., vitamin D deficiency, asteroid imbalance, prenatal screening) or to evaluate the risk ofdeveloping such a disorder based on an excess or deficiency of ananalyte of interest. For example, vitamin D metabolites are involved inmany important biological processes. Thus, abnormal increases ordecreases in their levels can alter biological functions within anorganism. A deficiency in vitamin D metabolites is associated withseveral human diseases including rickets, osteomalacia, osteoporosis,hypocalcemia, and hyperparathyroidism. An excess of vitamin Dmetabolites can be associated with hypercalcemia. Levels of circulatingvitamin D metabolites have been associated with the potential fordeveloping certain types of cancer. Higher serum 25-hydroxyvitamin D3levels have been associated with a decreased colorectal adenoma risk.See Peters et al., Cancer Epidemiology Biomarkers & Prevention,13:546-552 (2004). Similarly, disruption of thehypothalamus-pituitary-adrenal axis can result in steroid imbalance andvarious disease states. For example, dysfunction of the adrenal glandsis associated with cortisol and aldosterol deficiency. Decreasedcortisol secretion from the adrenal is the major cause of congenitaladrenal hyperplasia. A deficiency in the production of sex steroids suchas estrogen or testosterone can result in the loss of secondary sexualcharacteristics. Alterations in sex steroids can be indicative ofdisorders including cancers, and natural processes such as onset ofmenopause. In some cases, the methods provided herein can be used todetermine whether or not a mammal (e.g., a human individual) has ahealth state associated with altered levels of an analyte. Inparticular, levels of certain analytes can serve as biochemicalindicators of a disorder, regardless of whether physiologic orbehavioral symptoms of the disorder are manifest in the individual.

The methods described herein are useful for determining therapeuticefficacy of a particular treatment. When a treatment is selected andtreatment starts, the individual can be monitored periodically bycollecting biological samples at two or more intervals, measuring theexpression level of an analyte corresponding to a given time intervalpre- and post-treatment, and comparing expression levels over time. Onthe basis of any trends observed with respect to increasing, decreasing,or stabilizing expression levels, a clinician or other health-careprofessional may choose to continue treatment as is, to discontinuetreatment, or to adjust the treatment plan with the goal of seeingimprovement over time. In some cases, the individual can be offeredadditional or alternative therapeutic options. In some cases, changes inthe level of a particular analyte can indicate compliance ornon-compliance with a particular treatment plan. For example, lower thanexpected serum levels of 25-hydroxyvitamin D2 can indicate theindividual's non-compliance with or poor response to a therapeuticregimen of vitamin D supplements. Therefore, the methods and materialsprovided herein are applicable to screening, diagnosis, prognosis,monitoring therapy and compliance, and any other application in whichdetermining the amount of an analyte, such as a vitamin D metabolite, isuseful.

Information collected according to the methods provided herein can becommunicated to another person. For example, a researcher ordiagnostician can communicate such information to a clinician or othermedical professional. Any appropriate method can be used to communicateanalyte level information to another person (e.g., a professional), andinformation can be communicated directly or indirectly. For example, alaboratory technician can input analyte level information into acomputer-based record. In some cases, information can be communicated bymaking a physical alteration to medical or research records. Forexample, a medical professional can make a permanent notation or flag amedical record for communicating a diagnosis to other health-careprofessionals reviewing the record. Any type of communication can beused (e.g., mail, e-mail, telephone, and face-to-face interactions).Information also can be communicated to a professional by making thatinformation electronically available to the professional. For example,information can be placed on a computer database such that a health-careprofessional can access the information. In addition, information can becommunicated to a hospital, clinic, or research facility serving as anagent for the professional.

Also provided herein are kits useful for determining the amount of ananalyte in a test sample, as is described herein. Typically, a kitincludes an isotopically-labeled calibrator standard and anisotopically-labeled internal standard, wherein the calibrator andinternal standards can be distinguished using a mass spectrometrytechnique. For example, an exemplary calibrator and internal standardpair can be differently isotopically-labeled forms of the same analytederivative, while another exemplary pair can be different analytederivatives with the same isotopic labels. More than one type ofisotopic label can be present in a calibrator and/or internal standard.In some embodiments, the isotopically-labeled standard is a biologicalmaterial. In another embodiment, the kit can include anisotopically-labeled calibrator standard and an isotopically-labeledinternal standard as described herein and a label that indicates thatthe contents are to be used for determining an amount of an analyte in atest sample. For determining the amount of a selected analyte theisotoptically-labeled internal standard and isotopically-labeledcalibrator standard can be differently isotopically-labeled forms of theanalyte that differ sufficiently to allow their individual detectionusing a mass spectrometry technique. Specific isotopically-labeledcompounds useful as internal standards and calibrator standards areshown in FIG. 3.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description herein. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1

This example describes use of calibration curves prepared usingistopically labeled calibrator standards to accurately quantitate theamount of vitamin D analogs in quality-control samples by a massspectrometry technique.

In order to test performance of an assay for detecting vitamin Dmetabolites using the isotope-labeled vitamin D standard compoundsdescribed herein, the assay detailed below was used to quantify theamount of total vitamin D (the sum of the concentrations of25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) in 5 samples provided bythe vitamin D External Quality Assessment Scheme (DEQAS) (deqas.org onthe World Wide Web). The DEQAS scheme regularly distributes pooled humanserum samples to participating laboratories that analyze the25-hydroxyvitamin D content of the samples and report the results backto the scheme. There are typically 400+laboratories participating in thescheme employing 13 or more different analytical techniques. Theresulting means for each analysis type and the overall “trimmedlaboratory mean” is reported back to the participants.

DEQAS samples were quantified against a calibration curve of serumcalibrators spiked with deuterated 25-hydroxyvitamin D2 and25-hydroxyvitamin D3. The results described below showed good agreementbetween the results obtained using the labeled controls and calibratorsand the results from other laboratories participating in the scheme (seeTable 1).

TABLE 1 Results from DEQAS proficiency testing samples and comparisonwith data from other laboratories. Results from DEQAS study PKI Diasorinmeasured LCMS Liason DEQAS Total D mean mean All Lab Sample (ng/mL)(ng/mL) (ng/mL) mean 1 13.163 12.6 13.2 13.4 2 36.576 37.5 28.2 30.4 39.821 10.1 9.1 10.1 4 18.552 19.5 18.8 18.9 5 43.302 42.6 29.5 28.8

Calibration curves were constructed from the vitamin D calibratorsdescribed below and shown in Table 2. Actual spiked concentrations weredetermined by value assignment from a neat solution calibration curve ofthe labeled analytes. Analyte/internal standard ratios obtained from theDEQAS samples were compared with the kit calibration curves to determinethe concentration of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3(25OHD3) in the samples. The resulting concentrations were summed toobtain the “total D” concentration for comparison with the DEQAS data.Calibration curves are shown in FIG. 1A for 25-hydroxyvitamin D3(25OHD3) and 2A for 25-hydroxyvitamin D2 (25OHD2). The data reflectaverage signal ratio (signal intensity of calibrator and signalintensity of internal standard) with ±1 standard deviation error bars v.concentration of calibrator.

Production of labeled calibrators was performed as follows: Heavycharcoal stripped serum was spiked with 25-hydroxyvitamin-D2(26,26,26,27,27,27-d6) and 25-hydroxyvitamin-D3 (26,26,26,27,27,27-d6)to produce the following series of calibrators:

TABLE 2 Vitamin D Calibrator Levels. d6-25OHD2 d6-25OHD3 Level (ng/mL)(ng/mL) 1 0.64 0.63 2 1.27 1.26 3 6.55 6.47 4 15.91 15.71 5 28.40 28.066 65.55 64.75 7 159.05 157.11 8 216.31 213.67

Calibrators were aliquoted into 3 mL amber vials (2.05 mL per vial). Thecalibrators were then lyophilized and frozen in order to stabilize themfor subsequent use in the assay.

Lyophilized calibrators were prepared for quantitation by removing vialsfrom −20° C. storage and thawing the serum vials to room temperature.The calibrators were then re-suspended in 2 mL of MS Grade Water, andplaced on rocker for 1 hour, rotating after 30 minutes.

4-Phenyl-1,2,4-Triazoline-3,5-dione (PTAD)was prepared by weighing outabout 30 mg PTAD into an amber vial, and dissolving in MS GradeAcetonitrile to a final concentration of 1.5 mg/mL.

To prepare the Daily Working Solution (DWS), specific amounts ofinternal standards, 25-hydroxyvitamin-D2 (6,19,19-d3) and25-hydroxyvitamin-D3 (6,19,19-d3) were dissolved in acetonitrile with0.1% formic acid to achieve a final concentration of 30 ng/mL.

The assay preparation was performed as follows (the procedure wasfollowed to process test samples and calibrators):

-   1) 200 μL DWS was added to each well of a 500 μL Axygen Assay Plate.-   2) 100 μL of the appropriate Serum Sample was added to each well.-   3) The plate was covered with a SSP Plate Mat.-   4) The plate was shaken at 25° C. at 750 rpm for 10 minutes on    Wallac NCS Incubator.-   5) The plate was introduced to a Hettich Rotanta 460R Centrifuge and    process at 4600 rpm at 25° C. for 30 minutes.-   6) The plate was carefully removed from the centrifuge and the cover    was carefully removed.-   7) 150 μL of the sample mixture was transfered from each well to the    corresponding wells of a 350 μL Nunc Plate.-   8) The sample was evaporated to dryness on Evaporex EVX-192 Plate    Concentrator (Upper Temperature 45° C., Lower Temperature 80° C.).    Total drying time is about 15 minutes.-   9) 40 μL of PTAD solution was added to each well containing sample    mixture.-   10) The plate was covered with an Adhesive Microplate Cover and    Shaken at 25° C. at 750 rpm for 10 minutes on Wallac NCS Incubator.-   11) After incubation was complete, the derivatization reaction was    quenched by adding 40 μL of 0.1% Formic Acid to each well.-   12) The plate was covered with Aluminum Foil and shaken at 25° C. at    750 rpm for 10 min on Wallac NCS Incubator.-   14) Processed samples were analyzed on a Waters Quattro Micro triple    quadrupole.

Example 2

In order to test performance of an assay for detecting vitamin Dmetabolites using the isotope-labeled vitamin D calibrators describedherein, the assay detailed below was used to quantify the amount of25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in 3 serum based samplesprovided by National Institute of Standards and Technology (NIST,nist.gov on the World Wide Web).

NIST standard reference material (SRM) 972 consists of multiple levelsof serum based certified standards. These levels are prepared by NISTfrom either human serum or human/horse sera combinations. NIST SRM972level 3 is enriched with specific levels of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3. With exception of 25-hydroxyvitamin D2 for level1, all of the other levels are certified by NIST.

NIST SRM samples were quantified against a calibration curve of serumcalibrators spiked with 25-hydroxyvitamin-D2 (26,26,26,27,27,27-d6) and25-hydroxyvitamin-D3 (26,26,26,27,27,27-d6). Calibration curves wereconstructed from the vitamin D calibrators described below and shown inTable 3. Actual spiked concentrations were determined by valueassignment from a neat solution calibration curve of the labeledanalytes. Calibration curves are shown in FIGS. 1B and 2B. The datareflect average signal ratio (signal intensity of calibrator and signalintensity of internal standard) with ±1 standard deviation error bars v.concentration of calibrator.

TABLE 3 Vitamin D Calibrator Levels. d6-25OHD2 d6-25OHD3 Level (ng/mL)(ng/mL) 1 3.71 3.28 2 7.04 6.51 3 16.32 14.28 4 32.04 29.09 5 59.5259.22 6 124.95 120.80

Analyte/internal standard ratios obtained from the NIST samples werecompared with the calibration curves to determine the concentration of25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the samples.

The results described below showed good agreement between the resultsobtained using the labeled controls and calibrators and the NISTreference and certified values (Table 4).

TABLE 4 Results from NIST SRM testing samples and comparison withreference and certified values NIST certified or PKI measured referencevalue (ng/mL) (ng/ml) NIST 25OH 25OH 25OH Sample D2 D3 25OH D2 D3 1 0.5024.30 0.6* 23.9 2 1.81 13.22 1.71 12.3 3 26.64 19.73 26.4 18.5 *NISTreference value. the rest are NIST certified values.

Production of labeled calibrators was performed as described inExample 1. Serum sample preparation was performed as described inExample 1. The samples were analyzed on a Waters TQD MSMS detectoremploying Luna 3 μm C8(2) 100 Å 50×3.0 mm HPLC column.

Example 3

In order to test performance of an assay for detecting vitamin Dmetabolites using the isotope-labeled vitamin D standard calibrators asdescribed herein, the assay detailed below was performed to quantify theamount of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in threeartificially enriched serum samples.

Production of artificially enriched serum test samples was performed asfollows: off-the-clot serum was spiked with 25-hydroxyvitamin D2(25OHD2) and 25-hydroxyvitamin D3 (25OHD3) to produce the followingseries of enriched samples:

TABLE 4 Vitamin D enriched samples and enrichment degree (ng/ml). 25OHD225OHD3 Level (ng/mL) (ng/mL) 1 11.9 0.0 2 23.7 0.0 3 36.6 0.0 4 49.5 5.15 69.3 10.1 6 89.1 30.0

Aliquots of the samples were placed into 5-mL amber vials (2.05 mL pervial) and frozen to stabilize them for subsequent use in the assay.

The enriched samples were quantified against a calibration curve ofserum calibrators spiked with 25-hydroxyvitamin-D2(26,26,26,27,27,27-d6) (d6-25OHD2) and 25-hydroxyvitamin-D3(26,26,26,27,27,27-d6) (d6-25OHD3). Calibration curves were constructedfrom the vitamin D calibrators described below and shown in Table 5.

TABLE 5 Vitamin D Calibrator Levels. d6-25OHD2 d6-25OHD3 Level (ng/mL)(ng/mL) 1 3.67 3.51 2 6.55 6.80 3 15.21 15.62 4 28.19 27.64 5 61.9357.21 6 119.99 111.30

Analyte/internal standard ratios obtained from the enriched samples werecompared with the kit calibration curves to determine the concentrationof 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the samples.Examples of calibration curves for 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 are shown in FIGS. 2B and 1B, respectively.

The results described below showed good analyte recoveries (i.e. closeto 100%) from the quantitation using the calibrators described herein(Table 6)

TABLE 6 Analyte recovery results (%) from enriched samples 25OHD2 25OHD3endogenous PKI PKI % and measured Spiked % measured Spiked Re- enrichedConc. Conc. Recov- Conc. Conc. cov- samples (ng/ml) (ng/mL) ery (ng/ml)(ng/mL) ery endogenous 1.5 0.0 NA 28.1 0.0 NA 1 13.5 11.9 102% 27.1 0.0NA 2 24.7 23.7  98% 25.5 0.0 NA 3 40.4 36.6 106% 32.9 5.1  94% 4 54.749.5 107% 38.9 10.1 107% 5 71.3 69.3 101% 61.0 30.0 110% 6 95.2 89.1105% 81.2 49.8 106%Production of labeled calibrators was performed as described in example2.Serum sample preparation and analysis was performed as described inExample 2.

Example 4

Quantitation of such analytes such as steroids and vitamin D metabolitesis often performed using calibrator samples prepared using a biologicalmatrix, such as serum. In order to quantitate clinical samples, thematrix based calibrators contain quantities of calibrators that bracketthe clinically relevant concentration range. Typically, the calibratorsamples are produced by first removing the endogenous analytes by eithercharcoal or resin stripping, followed by adding specific amounts ofcalibrator. However, the process of charcoal stripping is not alwaysefficient. FIG. 4 illustrates the inefficiency phenomenon byillustrating the mass spectrum data for various samples after charcoalstripping: testosterone (A), progesterone (B), and 25-hydroxyvitamin D3(C). In each of these samples, the respective analytes remain atappreciably higher levels than desirable for quantitation of clinicalsamples (lower clinical range for testosterone, progesterone and25-hydroxyvitamin D3 (25OHD3) are 0.03 ng/ml, 0.08 ng/ml and 5 ng/ml,respectively). Therefore, as described above, employing isotopicallylabeled standards to produce sample matrix based calibrators overcomesthis inefficiency.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for determining an amount of an analyte in a test sample,the method comprising: (a) providing a set of calibrator samples, eachcalibrator sample comprising a different amount of a firstisotopically-labeled standard in a biological matrix, (b) providing toeach calibrator sample a selected constant amount of a secondisotopically labeled standard, the first and second isotopically labeledstandards having distinct isotope content; (c) subjecting the calibratorsamples comprising the first and second isotopically-labeled standardsto mass spectrometry to determine signal intensities corresponding tothe first and second isotopically-labeled standards; (d) generating acalibration curve based on ratios of the determined signal intensitiesof the first and second isotopically-labeled standards as a function ofthe amount of the first isotopically-labeled standard in the calibratorsamples; (e) contacting a test sample with the selected constant amountof the second isotopically-labeled standard; (f) subjecting the testsample comprising the second isotopically-labeled standard to massspectrometry to determine signal intensities corresponding to the secondisotopically labeled standard and the analyte, and (g) using thecalibration curve and the determined signal intensities corresponding tothe second isotopically labeled standard and the analyte to determinethe amount of the analyte in the test sample.
 2. The method of claim 1,wherein the analyte is selected from a secosteroid metabolite and asteroid metabolite.
 3. The method of claim 2, wherein the secosteroidmetabolite is selected from 25-hydroxyvitamin D3, 1-hydroxyvitamin D3,1, 25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, 1α-hydroxyvitamin D2,and 24-hydroxyvitamin D2.
 4. The method of claim 1, wherein the analyteis a vitamin D3 metabolite and the first isotopically-labeled standardis selected from isotopically labeled 1, 25-dihydroxyvitamin D3 andisotopically-labeled 25-hydroxyvitamin-D3.
 5. The method of claim 1,wherein the first isotopically-labeled standard is labeled with ²H, ¹³C,¹⁵N, ¹⁸O, or a combination thereof.
 6. The method of claim 5, whereinthe first isotopically-labeled standard is selected from labeled1α,25-dihydroxyvitamin D3, d₆-labeled 25-hydroxyvitamin-D3, d₃-labeled1, 25-dihydroxyvitamin D3 and d₃-labeled 25-hydroxyvitamin-D3.
 7. Themethod of claim 6, wherein the first isotopically-labeled standard is25-hydroxyvitamin-D3 (26,26,26,27,27,27-d6).
 8. The method of claim 1,wherein the analyte is a vitamin D3 metabolite and the secondisotopically-labeled standard is selected from isotopically labeled 1,25-dihydroxyvitamin D3 and isotopically-labeled 25-hydroxyvitamin-D3. 9.The method of claim 1, wherein the second isotopically-labeled standardis labeled with deuterium.
 10. The method of claim 9, wherein the secondisotopically-labeled standard is selected from d₆-labeled 1,25-dihydroxyvitamin D3, d₆-labeled 25-hydroxyvitamin-D3, d₃-labeled 1,25-dihydroxyvitamin D3 and d₃-labeled 25-hydroxyvitamin-D3.
 11. Themethod of claim 10, wherein the second isotopically-labeled standard is25-hydroxyvitamin-D3 (6,19,19-d3).
 12. The method of claim 1, whereinthe analyte is a vitamin D2 metabolite.
 13. The method of claim 12,wherein the analyte is a vitamin D2 metabolite and the firstisotopically-labeled standard is selected from 1,25-dihydroxyvitamin D2and 25-hydroxyvitamin-D2.
 14. The method of claim 5, wherein the firstisotopically-labeled standard is 25-hydroxyvitamin-D2(26,26,26,27,27,27-d6).
 15. The method of claim 12, wherein the analyteis a vitamin D2 metabolite and the second isotopically-labeled standardis selected from 1,25-dihydroxyvitamin D2 and 25-hydroxyvitamin-D2. 16.The method of claim 15, wherein the second isotopically-labeled standardis 25-hydroxyvitamin-D2 (6,19,19-d3).
 17. The method of claim 1, whereinthe analyte is a steroid selected from the group consisting of:11-deoxycortisol, 21-deoxycortisol, 17-α-hydroxyprogesterone,aldosterone, 4-androstene-3,17-dione, corticosterone,deoxycorticosterone, cortisol, dehydroepiandrosterone,dehydroepiandrosterone sulfate, estradiol, estriol, estrone,progesterone, 5-α-dihydrotestosterone, testosterone, pregnenolone,17-α-hydroxypregnenolone, 5-α-androstane-3b,17b-diol,2-hydroxyestradiol, 4-hydroxyestradiol, high-density lipoprotein (HDL)cholesterol, low-density lipoprotein (LDL) cholesterol, and verylow-density lipoprotein (VLDL) cholesterol, and the first and secondisotopically labeled standards are isotopically-labeled analogs of theselected analyte.
 18. The method of claim 5, wherein the analyte istestosterone and an isotopically-labeled standard is testosterone(3,4-¹³C; 2,2,4,6,6-d5).
 19. The method of claim 5, wherein the analyteis 4-androstene-3,17-dione and an isotopically-labeled standard is4-androstene-3,17-dione (3,4-¹³C; 2,2,4,6,6-d5).
 20. The method of claim5, wherein the analyte is dehydroepiandrosterone and anisotopically-labeled standard is dehydroepiandrosterone (3,4-¹³C;2,2,3,4,4,6-d5).
 21. The method of claim 5, wherein the analyte isprogesterone and the isotopically-labeled standard is progesterone(3,4-¹³C; 2,2,5,6,6,17a,21,21,21-d9).
 22. The method of claim 1, whereinthe mass spectrometry comprises a technique selected from LC-MS andLC-MS/MS.
 23. The method of claim 1, wherein the test sample is abiological sample.
 24. The method of claim 23, wherein the biologicalsample is selected from the group consisting of blood, serum, plasma,saliva, and urine.
 25. The method of claim 24, wherein the blood isprovided dried on a paper matrix.
 26. The method of claim 1, whereinfirst and second isotopically-labeled standards have distinct isotopecontents.
 27. The method of claim 1, wherein first and secondisotopically-labeled standards each comprise a different number ofisotopic labels comprising the same isotope.
 28. A kit for determiningan amount of an analyte in a test sample, comprising: an isotopicallylabeled calibrator standard, and an isotopically labeled internalstandard, wherein the calibrator and internal standards have distinctisotope content.